high-end chlorophyll fluorescence analysis with the multi

Heinz Walz GmbH, Effeltrich, Germany http://www.walz.com/ PAM Application Notes (2011) 1: 1 - 21

MC-PAM. Various light qualities. 1

High-end chlorophyll fluorescence analysis with the MULTI-COLOR-PAM. I. Various light qualities and their applications. Ulrich Schreiber, Christof Klughammer and Jörg Kolbowski

Abstract The MULTI-COLOR-PAM Multiple Excitation Wavelength Chlorophyll Fluorescence Analyzer is unique in provid-ing 6 different wavelengths of pulse-modulated measuring light, ML (400, 440, 480, 540, 590 and 625 nm), as well as 6 different wavelengths of actinic light, namely 440, 480, 540, 590, 625 and 420-640 nm (white). The variously col-ored actinic light can be used for continuous illumination (AL), maximal intensity single turnover pulses (ST), high intensity multiple turnover pulses (MT) and Saturation Pulses (SP). In addition, far-red light (FR, peaking at 725 nm) is provided for preferential excitation of PS I. This article outlines the properties and applications of the various colors of measuring and actinic light provided by the MULTI-COLOR-PAM. The pulse-modulated ML can be applied with vastly different pulse frequencies ranging between 10 to 200000 Hz, thus enabling continuous monitoring of quasi-dark fluorescence yield (Fo) as well as measurement of fast induction kinetics in the sub-ms time range using the same ML of various colors. Analysis of the O-I1 fluorescence rise kinetics in saturating light allows determination of the wavelength and sample specific functional absorption cross-section of PS II, Sigma(II)λ, with which the PS II turnover rate at a given incident PAR can be calculated. Vastly different light response curves can be obtained with AL of different colors. Based on Sigma(II)λ the usual PAR, in units of µmol quanta/(m² · s), can be converted into PAR(II), in units of PS II effective quanta/s, in order to compare the responses obtained with differently colored AL. A fluorescence-based electron transport rate ETR(II) = PAR(II) · Y(II)/Y(II)max is defined, which in contrast to the usual relative ETR can describe the rate of electron transport even in dilute suspensions of unicellular algae and cyanobacteria. When chlorophyll content is known, an absolute rate of O2 evolution can be estimated from ETR(II). The MULTI-COLOR-PAM is also well suited for measurements with leaves, for which a special optical unit with clip-holder is provided. For this application the use of 440 nm ML/AL/ST/MT/SP and detection of F < 710 nm are recommended.

1 Introduction

1.1 Unique features of the MULTI-COLOR-PAM The MULTI-COLOR-PAM is founded on the tradition of previous Pulse-Amplitude-Modulation (PAM) chloro-phyll fluorometers, i.e. mainly the PAM-100, XE-PAM, PHYTO-PAM, DUAL-PAM-100 and PAM-2500. It now combines virtues of these proven instruments in one compact device and in addition offers a number of unique properties, rendering it the high-end choice for chloro-phyll fluorescence analysis. It is not only suited for reli-able routine measurements, like assessment of effective PS II quantum yield, non-photochemical quenching and relative electron transport rate, but also for sophisticated work on special aspects of photosynthesis, like wave-length dependence of electron transport rate, reversible state 1-state 2 transitions and changes of absorption cross-section of PS II.

At the core of the MULTI-COLOR-PAM is a multi-color Chip-on-Board (COB) LED Array specially developed for this particular purpose in cooperation with PerkinEl-mer Elcos GmbH (Pfaffenhofen, Germany) and a dedi-cated microprocessor-based controller for its light-output and the processing of the fluorescence responses. Special

user friendly software is provided for recording and ana-lyzing the data.

The multi-color COB-Array consists of 60 Power-LED-chips on a 10 x 10 mm area, featuring a total of 8 differ-ent colors, which are randomly mixed by a 10 x 10 mm Perspex light pipe. This leads to a 10 x 10 mm glass cu-vette in the optical unit, in which the suspended sample can be stirred continuously. For leaf measurements an op-tional optical unit is provided, featuring a pyramidal Per-spex light pipe (base 10 x 10 mm, top 6 x 6 mm) and a clip-holder.

In conjunction with the dedicated user-software, this set-up provides the means for creating a unique variety of different light qualities in terms of colors, intensities, pulse-forms and pulse-frequencies. Default settings and automated measuring routines are provided for standard measurements that have proven useful for basic chloro-phyll fluorescence analysis. For advanced studies the MULTI-COLOR-PAM offers numerous possibilities of user-defined illumination protocols. In this respect, the MULTI-COLOR-PAM resembles a sophisticated music



instrument, like an organ with numerous “registrations”, the performance of which depends on the skill and imagination of the player.

The MULTI-COLOR-PAM is unique in combining the information obtained from fast kinetic measurements in the sub-ms time range with steady-state information ob-tained e.g. with the help of light response curves. In par-ticular, reliable assessment of the wavelength-dependent absorption cross-section of PS II, Sigma (II), opens the way for profound fluorescence-based analysis of photo-synthetic electron transport and its regulation in variously pigmented organisms.

The many different aspects and applications of the MULTI-COLOR-PAM will be outlined in a series of PAM Application Note (PAN) articles. The present arti-cle deals with the different light qualities and their appli-cations. A separate article will be dedicated to far-red light, of which a wide range of intensities is provided for preferential excitation of PS I. Particular emphasis is put on the fact that light responses of photosynthetic organ-isms essentially depend on light color. To take account of this color-specific behavior, the usual scale of wave-length-independent photosynthetically active radiation (PAR scale) needs to be transformed into a PS II-specific, wavelength-dependent “PAR(II) scale”. The MULTI-COLOR-PAM is unique in providing the means for trans-formation of the original PAR scale into a PAR(II) scale.

1.2 Pulse-modulated measuring light vs. actinic light

For understanding PAM fluorimetry in general and the MULTI-COLOR-PAM in particular, it is important to ra-tionalize the difference between measuring light (ML) and actinic light (AL). The ML is pulse-modulated, i.e. consisting of µs light pulses applied at defined frequen-cies (i.e. with defined periodical dark intervals), which excite corresponding fluorescence pulses that are selec-tively amplified by a synchronized amplifier. The AL is not pulse-modulated and, hence, neither the signal caused by the AL as such nor the AL-excited fluorescence are processed by the selective amplifier system.

Generally speaking, all types of light that change the state of the photosynthetic apparatus have an “actinic effect”. In principle, this is also true for the pulse-modulated ML, if this is applied at sufficiently high intensity and pulse frequency. The MULTI-COLOR-PAM provides very strong ML pulses which, depending on pulse frequency, can have practically no or a rather strong actinic effect (see section on ML below).

In addition, there are special types of actinic illumination, that are single and multiple turnover flashes, which rap-idly close PS II reaction centers. In the PAM literature, however, AL normally stands for continuous illumination that serves for driving a continuous photosynthetic elec-tron flow. “Saturating single turnover flashes” (ST) are applied to close all PS II reaction centers within a time shorter than the dark-reoxidation of the primary PS II ac-ceptor. “Multiple turnover flashes” (MT) are used to re-duce not only the primary acceptor QA, but also the sec-ondary plastoquinone (PQ) acceptor pool. “Saturating multiple turnover flashes”, also called “Saturation Pulses” (SP), have routinely been applied with PAM fluorimeters for SP-analysis of chlorophyll fluorescence, yielding a variety of fluorescence based parameters, in-cluding effective PS II quantum yield, Y(II), maximal PS II quantum yield, Fv/Fm = Y(II)max, and non-photochemical quenching, NPQ.

The MULTI-COLOR-PAM offers the possibility to choose between different colors of ML, AL, ST and MT/SP. This possibility is particularly important for work with organisms that display a particular antenna pigment composition or for the study of wavelength de-pendent processes, as e.g. reversible state transitions and orange carotenoid protein (OCP) quenching. While on first sight the color aspect may appear to apply only to algae and cyanobacteria, it is also important for leaf stud-ies. As recently emphasized by Rappaport et al. (2007), the actual fluorescence information from leaves is highly dependent on the depth of light penetration into the leaf, which again is determined by the light color.

A B C

Figure 1. Measuring light settings.



2 Properties of the different types of light provided by the MULTI-COLOR-PAM

2.1 Measuring light, ML

The MULTI-COLOR-PAM provides pulse-modulated ML with peak-wavelengths at 400 nm (dark-violet), 440 nm (blue-violet), 480 nm (cyan-blue), 540 nm (green), 590 nm (orange) and 625 nm (red-orange). Spu-rious ML at wavelengths above 640 nm is eliminated by a short-pass interference filter, so that the photodiode de-tector, which is protected by a long-pass filter (> 650 nm), will not see any ML directly, but only the pulse-modulated fluorescence (peak emission at 685 nm) that is excited by the ML.

An individual ML-pulse has a width of 1 µs, which even at maximal LED-current (i.e. maximal setting of ML-intensity) does not cause any appreciable closure of PS II reaction centers. In principle, this remains true also when ML-pulses are applied repetitively (pulse-modulation), as long as QA

- cannot accumulate, i.e. the dark-time between individual ML-pulses is sufficiently long for reoxidation of QA

- by the secondary PS II acceptor QB and the PQ-pool. There is, however, a type of variable fluorescence, which can be induced at extremely low light intensities, thus simulating a population of PS II centers inactive in secondary electron transport (Chylla et al. 1987, Lavergne and Leci 1993). The MULTI-COLOR-PAM is well-suited for studying the properties of such “inactive” PS II centers, which will be dealt with in a separate PAN communication.

For each ML color the user may choose between 20 dif-ferent intensity settings and 13 different pulse-frequency settings, which cover the range from 10 Hz to 100 kHz (i.e. factor of 104; cf. Fig. 1). For fast kinetics recordings, even a maximal pulse frequency (MF-max) of 200 kHz can be selected. A low frequency setting (MF-L) is used for quasi-dark assessment of Fo, i.e. of the minimal fluo-rescence yield observed when all PS II reaction centers are open. At the same time, a high frequency setting (MF-H) can be pre-selected, which becomes effective only when the actinic light is switched on and the “Auto MF-H function” is enabled. Hence, for the sake of a high

signal/noise ratio, relatively strong ML-pulses can be used, which at MF-L have a negligibly small actinic ef-fect. Upon automatically switching to MF-H, the light-induced changes of fluorescence yield are measured with high time resolution.

Measurements of relaxation kinetics (e.g. reflecting QA-

reoxidation following a saturating single turnover flash) demand on the one hand maximal MF for resolving the initial rate in the sub-ms time range and on the other hand low MF for resolving slow decay components under quasi-dark conditions. For this application a special “MF Log mode” is provided (under Fast Settings; cf. Fig. 1), which is initiated simultaneously with MF-max being turned off, resulting in logarithmically decreasing MF.

The relative intensities of the various ML colors differ considerably from each other. They are determined by the particular LED chip material and the number of chips on the COB-LED-array. At a given color, the effective ML-intensity is determined by the selected measuring light in-tensity and pulse-frequency.

At MF-H the contribution of the ML to overall actinic in-tensity can be considerable. The software accounts for this contribution when displaying the quantum flux den-sity of photosynthetically active radiation (PAR). Table 1 shows, for the 6 different ML-wavelengths, the number of chips on the COB-LED-Array and the PAR at the exit of the 10 x 10 mm Perspex light guide. The intensity val-ues show some variation between individual instruments, and for quantitative work they have to be measured with a suitable quantum sensor (like US-SQS/B or MQS-B, Walz).

The differences in PAR output of the variously colored chips are partially counterbalanced by the number of chips used. For example, the 480 nm ML features 3 chips only, as these chips are particularly powerful, whereas the 590 nm ML employs 8 chips, the light output of which is an order of magnitude lower than that of blue chips (cf. Table 1). The intensity of the 400 nm ML on purpose is much weaker than that of the other ML colors, as this ML wavelength is primarily provided for special applications like assessment of “yellow substances” (hu-mic acids, gilvin) in natural waters. The same multi-color-COB is also employed in the new PHYTO-PAM phytoplankton analyzer, where this aspect is quite impor-tant. The 480 nm ML displays the highest PAR. At 100 kHz and ML 10 the resulting 618 µmol quanta/(m² · s) is equivalent to relatively strong actinic light, which, e.g. in green algae, may even saturate photosynthetic electron flow. At 10 Hz, however, the time-integrated PAR only amounts to 0.0618 µmol quanta/(m² · s) , the actinic ef-fect of which can normally be neglected.

Table 1. Properties of the ML-part of the multi-color LED array. The PAR values apply for ML intensity setting 10 (ML10) and 100 kHz ML pulse frequency (MF 100K).

Peak wavelength, nm 400 440 480 540 590 625

Color

Number of LED chips on COB 1 4 3 8 8 6

PAR at output of Perspex rod ⎥⎦

⎤⎢⎣⎡

⋅μ

smE

2 26 468 618 403 198 318



Except for the 400 nm ML, the various colors of ML and AL, ST and MT/SP are generated by the same type of LED-chips. Therefore, when the same colors of ML and AL are selected, the actinic effects of both types of light are fully equivalent and correspondingly taken into ac-count by the software (e.g. in the display of light re-sponse curves).

2.2 Wavelength-dependence of normalized Fo/PAR and absorptance

Low ML-frequency (MF-L) is used to assess the quasi-dark level of fluorescence yield, Fo, the amplitude of which depends on the color of the ML as well as on the antenna pigment composition and content of the investi-gated organism.

In Table 2 and Fig. 2, the Fo values measured with 440, 480, 540, 590 and 625 nm ML in dilute suspensions of green algae (Chlorella vulgaris) and cyanobacteria (Synechocystis PCC 6403) are compared using identical settings of ML-intensity and Gain (signal amplification). The cell densities in the two suspensions were adjusted to give the same absorptance at 440 nm (see below). At the applied ML intensity setting (ML 5) and minimal pulse-frequency (MF-L 10), the effective intensities of the inci-dent PAR generally are too low to induce any fluores-cence increase beyond Fo (even with respect to “inactive PS II”).

Table 2. Comparison of Fo and PAR-scaled Fo (Fo/PAR) of dilute suspensions of Chlorella and Synechocystis measured with 5 different colors at identical settings of ML-intensity (ML 5) and minimal pulse-frequency (MF-L 10). The Fo/PAR values were normalized to give 1 rel. unit at 625 nm with Synechocystis, where the maximal signal was obtained.

Parameter Unit

Peak wavelength of ML nm 440 480 540 590 625

Incident PAR µmol m2·s 0.0234 0.0309 0.0201 0.0099 0.0159

Incident PAR rel. units 75.7 100.0 65.2 32.0 51.5

Fo(Chlorella)λ Volt 2.294 2.366 0.389 0.252 0.522

Fo(Chlorella)λ

PAR rel.

units 0.917 0.716 0.181 0.238 0.307

Fo(Synechocystis)λ Volt 0.359 0.198 0.616 0.703 1.702

Fo(Synechocystis)λ

PAR rel.

units 0.143 0.060 0.286 0.665 1.000

The relative PAR-values of the differently colored ML are important for interpretation of the Fo information in terms of PS II excitation. The PAR-scaled Fo levels (i.e. Fo/PAR) measured with various ML-colors provide the equivalent of an approximate 5-point fluorescence excita-tion spectrum (see Fig. 2). The Fo/PAR data were nor-malized to 1 relative unit at the maximal signal value, which was observed with Synechocystis and 625 nm exci-tation.

The wavelength-dependence of dark-fluorescence yield, Fo, differs considerably between Chlorella and Synecho-cystis. Despite the identical absorptance at 440 nm, i.e. although the same fraction of incident 440 nm quanta is absorbed in the Chlorella and Synechocystis suspensions, the Fo(Chlorella)440 exceeds the Fo(Synechocystis)440 by a factor of 2.294/0.359 = 6.4. In contrast, when 625 nm excitation is used with the same samples, which in cyanobacteria is strongly absorbed by phycocyanin, the Fo(Synechocystis)625 exceeds the Fo(Chlorella)625 by a factor of 1.702/0.522=3.3.

Figure 2. A: Comparison of normalized Fo/PAR in dilute suspensions of Chlorella and Synechocystis. The data were normalized to unity at maximal relative Fo/PAR, i.e. 625 nm with Synechocystis. B: Comparison of absorptance in the same suspensions of Chlorella and Synechocystis, the PAR-scaled Fo data of which are compared in A. Cell densities of the two suspensions were adjusted to give the same absorptance at 440 nm.



The Fo/PAR plots of Chlorella and Synechocystis in Fig. 2A can be compared with the corresponding absorptance spectra in Fig. 2B, measured with the MULTI-COLOR-PAM under identical optical conditions.

The MULTI-COLOR-PAM allows a determination of the wavelength-dependent absorptance of the same sample in which chlorophyll fluorescence is measured using the standard Optical Unit ED-101US. For this purpose, the detector-unit has to be moved from the 90° position (rela-tive to the emitter-unit) to the 180° position, which just takes a couple of minutes. The long-pass filter in front of the detector has to be exchanged against a suitable neutral density filter (or pin-hole diaphragm), so that the pulse-modulated transmittance signals can be measured both with the suspension medium, I1(medium), and with the sample I1(sample). The absorptance a (=1-transmittance) is calculated as follows:

)medium(I)sample(I1a

1

1−= (1)

While the spectra of Fo/PAR and absorptance resemble each other with Chlorella, they differ considerably in the case of Synechocystis. This is due to the fact that chloro-phyll fluorescence originates mainly from PS II and that the difference in pigment composition between PS I and PS II is much larger in Synechocystis than in Chlorella. In cyanobacteria, most of the Chl a is contained in the PS I antenna, whereas the phycobilisomes constitute the main PS II antenna system. Comparison of the Fo/PAR and absorptance data show that measurements of wave-length dependent fluorescence provides complementary information on PS II absorption, which cannot be ob-tained from absorptance data, as besides PS I pigments also other pigments and constituents contribute to overall light absorption. As will be shown below, the most spe-cific information on PS II absorption is provided by measurements and analysis of the fast fluorescence rise upon onset of high intensity illumination of different col-ors (see section 3.2).

2.3 Actinic light, AL The MULTI-COLOR-PAM provides continuous AL with the same wavelengths as provided for ML, except for the 400 nm AL (see Table 1 and text above). In addition, also white actinic light (420-640 nm) is provided. Wave-lengths above 640 nm are eliminated by a short-pass in-terference filter, so that the photodiode detector, which is protected by a long-pass filter (> 650 nm), does not see any AL directly, but only the non-modulated fluores-cence (peak emission at 685 nm) that is excited by AL. Non-modulated fluorescence, however, is not processed by the PAM amplifier system, which selectively ampli-fies the pulse-modulated signal. Actinic light generally serves for driving photosynthetic electron transport by charge separation at the reaction centers of PS I and PS II. As already mentioned above, in PAM applications the term AL is mostly used for continuous actinic light,

whereas very short pulses (up to 50 µs width) of very strong actinic light are referred to as “single turnover flashes” (ST) and strong longer pulses (up to about 800 ms) are called “multiple turnover flashes” (MT) or “saturation pulses” (SP). These special types of actinic il-lumination will be dealt with in separate sections below. The same applies to far-red actinic light (FR), which is preferentially absorbed by PS I, which will be dealt with in a separate PAN-article.

Figure 3. Upper panel: Screenshot of default PAR list for 440 nm. Left column, intensity settings. Center column, relative current val-ues. Right column, PAR values in µmol quanta/(m² · s). Lower panel: PAR values in µmol quanta/(m² · s) as a function of AL-intensity setting for one of six AL-colors (AL color #1, 440 nm) as defined by the default current/PAR list for suspensions (de-fault_MC.par).



For each color the user may choose between 20 intensity settings, corresponding to defined LED currents and PAR values at the exit of the Perspex light guide of the emitter unit. For standard applications default current/PAR lists with progressively increasing intensity settings are pro-vided (default_MC.par for suspensions and de-fault_leaf.par for leaves). The default lists can be readily modified by the user and saved in new par-files. LED current can be adjusted in 255 steps. The PAR defined in the default lists were measured with the spherical quan-tum sensor US-SQS/B and the planar sensor MQS-B for suspensions and leaves, respectively (Heinz Walz GmbH). These sensors can be directly connected to the Ext.Sensor input of the MULTI-COLOR-PAM Power-and-Control unit. A special routine is provided for auto-mated measurements of PAR-lists. Most importantly, the PAR-signals are processed with high time resolution, so that even the intensities of MT pulses in the ms time range are reliably assessed.

As already pointed out in the section on measuring light (ML), the ML may contribute significantly to overall PAR, if applied at high pulse frequency. Based on the given information on presently effective ML-intensity and ML-frequency settings, the corresponding PAR is calculated by the PamWin program and taken into ac-count in the PAR display.

In the PAR list, the PAR-value for AL-intensity setting 0 corresponds to ML of the same color at ML Int. setting 10 and MF 100 kHz. It is assumed that the PAR contrib-uted by the ML is proportional to ML-intensity and ML-frequency. The screenshot in Fig. 3 (top) shows the de-fault PAR-list for AL-color #1 (440 nm). In the bottom panel the corresponding plot of the PAR in µmol quanta/(m² · s) vs. the AL-intensity settings is displayed.

Table 3 shows for the 6 different AL-colors the number of chips on the COB-LED-Array and the PAR at the exit of the 10 x 10 mm Perspex light guide for the maximal AL intensity setting 20 (AL 20). In practice, the overall PAR is somewhat increased by the PAR contribution of the ML at high pulse-frequency, which is taken into ac-count. For all measurements the overall PAR calculated according to the current PAR list is saved together with the fluorescence data.

The PAR output per chip differs considerably for the various colors, which to some extent is balanced by the number of chips. While white AL yields the highest PAR, its role differs from that of the other colors, as no white ML is provided and, hence, depending on the choice of ML-color, ML-intensity and ML-frequency, the spectral composition of the overall actinic illumination will vary, when white AL is selected. With green organisms, in conjunction with white AL the use of green ML may be recommended, as this contributes only marginally to the overall actinic effect. The white LED chips contribute significantly to the overall intensity of saturating ST flashes (see section below), where spectral composition is not important, as long as saturation is reached.

While normally the same color is used for ML and AL, it is also possible to select different colors. For example, in the study of cyanobacteria, Chl a fluorescence can be di-rectly excited by weak 440 nm ML absorbed by Chl a in the thylakoid membrane, whereas electron transport can be driven by 625 nm AL absorbed within the phycobili-somes. In this way, important information on reversible state 1-state 2 transitions can be obtained. Examples of such measurements will be presented in a separate PAN article.

AL is used routinely for measurements of dark-light in-duction kinetics (Kautsky effect) and for light response curves (Light Curves). For these applications, the Pam-Win user surface provides the “Slow Kinetics”, “Fast Ki-netics” and “Light Curve” windows, as well as a number of automated measuring routines. In all these measure-ments the AL-induced responses depend not only on the PAR, but also on the color of the applied AL and the pigment composition of the investigated sample. Hence, for quantitative work, not only the knowledge of PAR is essential, but also of the wavelength-dependent func-tional cross-section of the investigated sample.

Figure 4. Screenshot of “Light Curve Edit” window. Left-most col-umn, illumination step of light treatment. Second column from left, PAR values in µmol quanta/(m² · s). Third column from left, intensity setting. Right-most column, width of illumination step (here 180 s for all steps).

Table 3. Properties of the AL-part of the multi-color LED array. The PAR values apply for maximal AL intensity setting 20 (AL 20).

Peak wavelength, nm 440 480 540 590 625 420-645

Color

Number of LED chips on COB 4 3 8 8 4 2

Maximum PAR at output of Perspex rod

⎥⎦⎤

⎢⎣⎡

⋅μ

smE

2 2807 3907 4724 1758 2250 4936



As in vivo fluorescence yield primarily reflects the effi-ciency of charge separation at PS II reaction centers, the absorption of AL by PS II is essential. It differs for vari-ous colors and organisms. For example, a given PAR of blue light will elicit a strong response in green algae, whereas the same light induces a weak response in

cyanobacteria. Conversely, a given PAR of short-wavelength red light (625 nm) may induce a strong effect in cyanobacteria, while a relatively weak response is ob-served in green algae. This aspect will be elaborated on in the following chapter 3.

3 Applications, calculations and definitions

3.1 Wavelength dependent Light Curves and rela-tive electron transport rates (ETR)

The light response of photosynthetic organisms routinely is analyzed with the help of fluorescence-based Light Curves (LC), consisting of a number of illumination steps with increasing intensities of photosynthetically active

radiation, PAR. The MULTI-COLOR-PAM is ideally suited for measuring LC with outstanding accuracy even at rather low chlorophyll content, with the advantage of avoiding light intensity gradients. The number of LC steps, as well as the widths and intensities of the individ-ual steps can be programmed by the user.

Figure 5. Typical Light Curves of Fm’ and of the derived complementary PS II quantum yields Y(II), Y(NPQ) and Y(NO) measured with dilute Chlorella suspension (300 µg Chl/L) using the MULTI-COLOR-PAM with 440 nm light.

Figure 6. Typical Light Curve of relative ETR measured with dilute Chlorella suspension (300 µg Chl/L) using the MULTI-COLOR-PAM with 440 nm light and a default ETR-factor of 0.42.



As the spectral properties of the ML are identical to those of the AL (provided the same color is selected), ML at high ML-Frequency (MF 2K etc.) can be used for cover-ing the low PAR range (see Fig. 4).

The illumination time periods (step widths) at the various PAR may vary between 10 s (resulting in so-called “rapid light curves”, RLC) and many minutes. The longer the il-lumination steps, the more fluorescence based LC ap-proach classical P vs. E curves (photosynthesis versus ir-radiance curves), where steady state is reached within each PAR-step, before photosynthetic rate is evaluated.

Figures 5 and 6 show screen shots of LC-data measured with a dilute suspension of Chlorella (300µg Chl/L) us-ing 440 nm light and 3 min illumination steps. In the ex-ample of Fig. 5 the fluorescence based complementary PS II quantum yields of photochemical energy conver-sion, Y(II), regulated non-photochemical energy conver-sion, Y(NPQ), and non-regulated non-photochemical en-ergy loss, Y(NO) are selected for display together with the original Fm’ data obtained with the help of Saturation Pulses (SP). These parameters provide basic information on the fate of absorbed light energy (Kramer et al. 2004, Klughammer and Schreiber 2008).

While a detailed discussion of the present data would be beyond the scope of the present application note, the pro-nounced biphasic PAR-dependence of Y(NPQ) may be pointed out, which to some extent also affects Y(II). The biphasic character does not disappear with longer illumi-nation steps (data not shown), i.e. it does not seem to be related to an induction effect. Based on the Y(II) data, relative electron transport data (rel. ETR) can be calcu-lated and plotted vs. PAR (see Fig. 6).

Relative electron transport rate (rel. ETR) as a fluores-cence-derived parameter was introduced for PAM-measurements with leaves (Schreiber et al. 1994):

factorETRPAR)II(YETR.rel −⋅⋅= (2)

(While formally units of µmol electrons/(m2 · s) apply for rel. ETR, in practice normally “rel. units” are used).

The ETR-factor is supposed to account for the fraction of overall incident PAR that is absorbed within PS II. In most published studies, however, no attempts were made to determine the ETR-factor, which simply has been as-sumed to correspond to that of a “model leaf”, with 50% of the PAR being distributed to PS II and 84% of the PAR being absorbed by photosynthetic pigments in a standard leaf (Björkman and Demmig 1987), so that normally a default ETR-factor of 0.42 is applied.

Without detailed knowledge of the true PS II specific ab-sorbance, ETR can give a rough estimate only of relative photosynthetic electron transport rate. In the case of di-lute algae suspensions, where a minor part of overall in-cident radiation is absorbed, normally rel. ETR is just treated as an intrinsic parameter of relative rate of PS II turnover without any information on absolute rate, calcu-lation of which would require information on PS II con-tent and the functional absorption cross-section of PS II (see sections 3.2 and 3.3).

Attempts of fluorescence-based determination of absolute rates of PS II turnover in algal suspensions so far have been the exception (see e.g. Gilbert et al. 2000a, Jakob et al. 2005). For calculation of absolute rates, the absorbed photosynthetic radiation (Qphar) has been estimated from algal absorption spectra and the emission spectrum of the applied light source (Gilbert et al. 2000b). This approach, however, is based on the overall absorption of the sam-ple, whereas for proper evaluation of fluorescence data the specific PS II absorption is decisive.

In the measurement of rel. ETR data presented in Fig. 6, no attempt was made to obtain absolute rates and the de-fault value of 0.42 was applied as ETR-factor. With this kind of approach, rel. ETR is independent of Chl content, just like Y(II), from which it is derived and, hence, essen-tially describes the relative frequency of charge separa-

Wavelength, nm α ETRmax Ik

440 0.292 23.3 79.8 480 0.277 31.6 114.1 540 0.262 134.8 515.3 590 0.250 73.9 295.7 625 0.275 58.9 214.2

Figure 7. ETRmax and Ik values of Chlorella plotted against the peak wavelength of the AL. Relative ETR light curves were measured with same Chlorella sample using different AL colors and a default ETR-factor of 0.42. Parameters were estimated according to the model of Eilers and Peeters (1988). The original relative ETR light curve with 440 nm AL is shown in Fig. 6. The table lists plotted data (ETRmax and Ik)and α.



tion at PS II reaction centers. Thus defined light curves of rel. ETR provide useful information, as long as the per-formance of a particular organism is studied using a fixed color of light, as is the case with standard PAM fluoro-meters. For this purpose, standard PAM-software pro-vides routines for fitting the LC-parameters α, ETRmax and Ik using models developed by Eilers and Peeters (1988) or Platt et al. (1980). The parameter α relates to the maximal PS II quantum yield (initial slope of LC). ETRmax is a measure of maximal relative rate and Ik re-lates to the PAR at which light saturation sets in (defined by ETRmax/α).

The values of these Light Curve parameters are strongly dependent on the color of light and PS II absorption of the sample. Hence, when LC are measured with the MULTI-COLOR-PAM using different AL-colors, vastly different ETRmax and Ik values are obtained for the same sample, (Fig. 7). Notably, in Chlorella there are even considerable differences between the two types of blue light (440 and 480 nm) and red light (625 nm). These data show that the same quantum flux density of differ-ently colored light within the range of “photosyntheti-cally active radiation”, PAR, can have vastly different outcomes, not only between differently pigmented organ-isms, but also within the same organism.

ETRmax and Ik display very similar wavelength depend-ency, in the case of Chlorella with peak and minimal val-ues at 440 and 540 nm, respectively. The ETRmax and Ik spectra resemble inverse Fo/PAR spectra (see Fig. 2). It should be kept in mind, however, that PS I contributes to Fo, and that ETRmax as well as Ik are not only dependent on PS II but also on PS I activity.

The MULTI-COLOR-PAM has opened the way for de-tailed studies of electron transport as a function of the color of radiation in photosynthetic organisms with largely different pigment composition. From the data in Fig. 7 it is obvious that in order to make full use of this new potential of the MULTI-COLOR-PAM, either a wavelength- and sample-dependent ETR-factor has to be defined or the quantum flux density of photosynthetically active radiation, PAR, has to be replaced by a PS II spe-cific quantum flux rate, PAR(II). The latter approach is advantageous, as it results in determination of an absolute rate, independent of chlorophyll content. It requires in-formation on the wavelength- and sample-dependent functional cross-section of PS II, Sigma(II)λ, which can be readily obtained with the MULTI-COLOR-PAM (see section 3.2). As will be outlined in section 3.3, PAR can be transformed into PAR(II) simply by multiplication with a Sigma(II)λ-based factor.

3.2 PAR and wavelength- and sample-dependent functional absorption cross-section of PS II, Sigma(II)λ

The PAR usually is defined for wavelengths between 400-700 nm (Sakshaugh et al. 1997) in units of µmol quanta/(m² · s). It is measured with calibrated quantum

sensors, like the spherical US-SQS/B (for suspensions) or the planar MQS-B (for leaves) from WALZ. Such de-vices measure the overall flux density of incident quanta, without making any distinction between quanta of differ-ent colors, as long as their wavelengths fall into the 400 to 700 nm PAR range.

Hence, the actual extent of PAR-absorption (whether by PS II or PS I or any other colored constituents) by the photosynthetically active sample normally is not taken into account. While this kind of approach may be feasible in the study of leaves, which display relatively flat ab-sorbance spectra and absorb most of the incident light, it does not work with dilute suspensions of unicellular al-gae and cyanobacteria, where PS II excitation by light of different wavelengths may vary by an order of magnitude (see data in section 2.2 above) and only a fraction of the incident light is absorbed.

Rappaport et al. (2007) recently pointed out that the “most commonly used unit for light intensity ... µmol of photons s-1 m-2 … has little experimental value since it cannot reliably be translated into a photochemical rate without knowing the absorbance of the sample, which is rarely the case”. Further, the authors note that “there is ... a real need for a more relevant unit which should be the number of electrons transferred per unit time and per PS II reaction center.” As will be outlined below, for quantitative work with the MULTI-COLOR-PAM, e.g. analysis of light response curves (LC), we propose the use of PAR(II) instead of PAR, which may serve the pur-pose suggested by these authors.

In PAM applications, exact information on PAR, meas-ured in units of µmol quanta/(m² · s) with standard quan-tum sensors is very essential, e.g. in conjunction with LC recordings. However, as already outlined in section 3.1, the PAR information has to be complemented with in-formation on the PS II efficiency of the applied PAR with respect to a given sample. Such information is contained in the wavelength-dependent functional cross-section of PS II, the Sigma(II)λ, which depends on both the spectral composition of the applied irradiance (i.e. the AL-color) and the PS II absorption properties of the investigated sample.

The value of Sigma(II)λ can be derived from the initial rise of fluorescence yield upon onset of saturating light intensity, which directly reflects the rate at which PS II centers are closed. The rate of charge separation of open PS II centers, k(II), matches the quantum flux rate of PS II effective light, which may be defined as PAR(II) (see section 3.3 below). In order to account for the over-lapping re-opening of PS II centers by secondary electron transport (reoxidation of QA

- by QB), either a PS II inhibi-tor like DCMU has to be added, which is not feasible for in vivo studies, or PAR(II) has to be extremely high, so that the reoxidation can be ignored (Kolber et al. 1998; Nedbal et al. 1999; Koblizek et al. 1998), or the rise ki-netics have to be corrected for the reoxidation rate.



The latter approach is applied with the MULTI-COLOR-PAM, which will be outlined in detail in a separate PAN article (Klughammer C, Kolbowski J and Schreiber U). Here just one practical example will be given using the same sample (dilute suspension of Chlorella) and AL-color (440 nm), with which the Light Curves in Figs. 5-6 were measured.

Figure 8 shows a typical example of the initial part of the increase of fluorescence yield induced by strong AL (in PAM-literature called O-I1 rise). The O-I1 rise basically corresponds to the O-J phase of the polyphasic OJIP ki-netics that have been described in detail by Strasser and co-workers (for reviews see Strasser et al. 2004, Stirbet and Govindjee 2011). There are, however, essential dif-ferences in the measuring techniques and definitions of I1 and J, which argue for different nomenclatures. The MULTI-COLOR-PAM allows the use of so-called “Fast Trigger Files” for routine measurements of fast kinetics. In the example of Figure 8, the pulse-modulated ML was triggered with 100 kHz pulse-frequency at 100 µs before onset of 440 nm AL.

At 1 ms after onset of AL, a saturating 50 µs multi-color single turnover pulse was applied (see section 4 on single turnover pulse, ST). The ST closes PS II reaction centers transiently so that the so-called I1-level of fluorescence yield can be determined. The I1-level corresponds to the maximal fluorescence yield that can be reached in the presence of an oxidized PQ-pool (for apparent PQ-quenching see Samson et al. 1999; Schreiber 2004).

Here, weak far-red (FR) background light is routinely ap-plied in order to assure a fully oxidized PQ-pool, which is particularly important in the study of algae and cyanobac-teria. Furthermore, FR-preillumination minimizes the

contribution of “inactive PS II” to the O-I1 kinetics. Pos-sible artifacts caused by rapid switching of high-intensity non-modulated light is avoided by corresponding gating time periods in the fast trigger files.

At a first approximation, assuming that the AL-driven in-crease of fluorescence yield is linearly correlated with accumulation of QA

-, and that the initial rise is negligibly slowed down by QA

- reoxidation, the kinetics can be de-scribed by a first order reaction, of which the time con-stant Tau = 1/k(II) corresponds to the time for reaching a QA-reduction level of 100(1-1/e) = 63.2% . When this approximation is applied to the O-I1 rise of Fig. 8, Tau = 0.379 ms is estimated. A very similar Tau-value (Tau = 0.382 ms) is obtained when the area growth method is applied (details on various fitting routines pro-vided by the MULTI-COLOR-PAM will be presented in an upcoming PAN article).

A thorough analysis of the O-I1 rise kinetics, however, has to take into account both QA

- reoxidation and non-linearity between ∆F and the fraction of reduced QA. This can be achieved by a fitting routine we have specially de-veloped for this purpose, which is based on the reversible radical pair model of PS II originally described by Lavergne and Trissl (1995) that was extended to take ac-count of QA

--reoxidation (Klughammer C, Kolbowski J and Schreiber U, in preparation).

Variable parameters in this model are

J Sigmoidicity parameter, which is closely re-lated to Joliot’s connectivity parameter, p

Tau Time constant of light-driven (by AL or MT) charge separation

Tau(reox) Time constant of QA- reoxidation.

Figure 8. Initial increase of fluorescence yield (O-I1 rise) in a dilute suspension of Chlorella (300 µg Chl/L) induced by 440 nm AL at intensity setting 18 (2131 µmol quanta/(m²·s)) in presence of far-red background light. Dashed yellow lines indicate Fo-level (O), assessed during a 50 µs period preceding onset of AL at time zero, and the I1-level that is determined with the help of a saturating single turnover pulse (ST) triggered 1 ms after onset of AL. The slope of the relaxation kinetics is extrapolated to the end of the 50 µs ST. The black line represents the O-I1 fit curve based on a PS II model which incorporates energy transfer between PS II units and reoxidation of the primary PS II acceptor QA (see text).



Directly measured parameters are the Fo and I1-levels, which define the total range of ∆F, that can be induced by a saturating ST in the presence of an oxidized PQ-pool

While determination of Tau and, hence, of k(II) = 1/Tau, does not require knowledge of PAR, the PAR has to be specified for calculation of Sigma(II)λ, the wavelength-dependent functional cross-section of PS II. Therefore, precise measurement of PAR under the optical conditions encountered by the sample is essential for accurate Sigma(II)λ determination, which again is a prerequisite for transformation of PAR into PAR(II) and calculation of absolute PS II turnover rates, ETR(II) (see below). For this purpose the PamWin software provides the special <Measure PAR Lists> routine for automated determina-tion of the PAR-values of AL, MT and MF-max for all colors offered by the Multi-Color-PAM.

For the O-I1 rise driven by 2131 µmol quanta/(m² · s) of 440 nm AL (displayed in Fig. 8), the following values were estimated by the O-I1 fit routine :

Tau = 0.173 ms k(II) = 1/Tau = 5.78 · 103 s-1

Tau(reox) = 0.340 ms J = 2.01 (corresponding to p = 0.67)

Sigma(II)440 = 4.51 nm2

With the help of these experimental data an example of step-by-step calculation of Sigma(II) may be illustrated, which in practice is carried out within fractions of a sec-ond by the PamWin software:

1) 1 µmol quanta/(m² · s) corresponds to 10-6 · 6.022· 1023 quanta/(m² · s) (where 6.022 · 1023 mol-1 is Avogadro’s constant), which is equivalent to 6.022 · 10-4 quanta · nm-2 · ms-1 (as 1 m2 corresponds to 1018 nm2).

2) 2131 µmol quanta/(m² · s) correspond to 1.283 quanta nm-2 · ms-1.

3) During the period of Tau = 0.173 ms (i.e. the experi-mentally determined PS II turnover time under the given conditions) 1.283 · 0.173 = 0.222 quanta nm-2 are absorbed.

4) Hence, 1/0.222 = 4.505 nm2 correspond to the func-tional PS II cross section in Chlorella for a 440 nm quantum, which is called Sigma(II)440. This cross-section may be visualized as the effective area of a PS II unit, exposed to a beam of photons, with the size of this area varying not only with pigment composi-tion, but also with the color of the incident light.

The above derived value of Sigma(II)440 specifically de-scribes the PS II efficiency of 440 nm AL for a dilute suspension of Chlorella under the standard condition of a non-energized sample with far-red background light. With a different AL color or another type of sample a dif-ferent Sigma(II) will apply, which can be readily deter-

mined by the MULTI-COLOR-PAM via another meas-urement of the O-I1 fluorescence rise followed by fitting of the time constant Tau and calculation of Sigma(II)λ.

Calculation of Sigma(II)λ by the MULTI-COLOR-PAM software is based on the following general equation:

PARLTau1

PARL)II(k)II(Sigma λ ⋅⋅

=⋅

=

(3)

where L is Avogadro’s constant, Tau is the time constant of PS II turnover during the O-I1 rise, PAR is the quan-tum flux density of the light driving the O-I1 rise and Sigma(II)λ is the wavelength and sample-dependent func-tional absorption cross-section of PS II.

In practice, the value of Sigma(II)λ is calculated by sub-stituting the general terms in the equation by the experi-mentally determined numbers with their corresponding units, e.g. in the case of the measurement of Fig. 8, with Tau = 0.173 ms and PAR = 2131 µmol/(m² · s):

)Chlorellain quanta nm 440 of absorptionfor II PSper (area nm 4.51

21316.0220.173nm10

1021316.0220.173m

smmol102131

mol106.022s100.173

1)II(Sigma

2

24

14

2

2

6233

440

=⋅⋅

=

⋅⋅⋅=

⋅⋅

⋅⋅

⋅⋅= −

−

This definition of Sigma(II)λ relates to the functional cross-section of PS II for the specific conditions under which the O-I1 rise measurement was carried out. Sigma(II)λ specifically applies for the reference state of a dark-adapted sample with open PS II reaction centers and oxidized PQ pool. It is not identical to the functional PS II cross-section, σPS II, defined in a less specific way (e.g. by Kolber et al. 1998) and, hence, can be modulated by changes in the PQ-redox state and various types of non-photochemical quenching.

Fig. 9 shows Sigma(II) values as a function of AL-color for a dilute suspension of Chlorella. O-I1 rise kinetics similar to the one shown in Fig. 8 were measured with the help of a pre-programmed script-file (Sigma1000Chlor_10.prg) for the five different AL-colors with 10 s time intervals between consecutive measurements. Continuous far-red background light was applied in order to keep the PQ-pool oxidized. Sigma(II)λ was determined by fitting the O-I1 rise curve as described for the curve in Fig. 8.

By comparing the Sigma(II) and Fo/PAR spectra for Chlorella in Fig. 9 and Fig. 2, respectively, it is apparent that Sigma(II) and Fo/PAR carry very similar information on PS II absorption of a sample. An important difference, however, is that Sigma(II) gives absolute information on the functional cross-section of PS II, which is independ-ent of Chl content, whereas Fo/PAR is proportional to



both Chl content and functional cross-section of PS II. Furthermore Fo/PAR depends on ML-intensity and gain parameters, which have no influence on Sigma(II), as measured with the MULTI-COLOR-PAM.

3.3 Definition of PAR(II) and ETR(II) The wavelength-dependent PS II-effective quantum flux rate is directly reflected in the k(II) determined by fitting the O-I1 rise kinetics measured at high PAR. There is di-rect correspondence between the PS II turnover rate, k(II), in units of electrons/(PS II · s) and the quantum ab-sorption rate at PS II reaction centers in units of quanta/(PS II · s). We propose the name PAR(II) for the latter, with the general definition derived from equation 3 above:

PARL)II(Sigma)II(k)II(PAR λ ⋅⋅==

(4)

where k(II) is the rate constant of PS II turnover, Sigma(II)λ is the functional cross-section of PS II, L is Avogadro’s constant, PAR is the quantum flux density of illumination and PAR(II) is the rate of quantum absorp-tion in PS II. PAR(II) is expressed in units of quanta/(PS II · s).

In practice, PAR(II) is calculated by substituting the gen-eral terms in this equation by the experimentally deter-mined values with their corresponding units (see example below). Most importantly, once Sigma(II) has been de-termined for one particular color and sample (via meas-urement of the O-I1 rise kinetics at a defined high light intensity), k(II) and PAR(II) can be derived for any other PAR, without any further measurements of fast kinetics.

An essential difference between PAR(II) and PAR is that

the former relates to the quantum absorption rate of PS II, which is independent of Chl content, whereas the latter represents a quantum flux density, from which a PS II quantum absorption rate can be calculated only, if the PS II content is known.

Consequently, based on PAR(II), also a wavelength- and sample-dependent ETR(II) can be defined:

Figure 9. Functional cross-section of PS II, Sigma(II) as a function of AL-color in a dilute suspension of Chlorella (300 µg Chl/L) derived from automated measurement of 5 consecutive O-I1 rise curves (Script-file Sigma1000Chlor_10.prg) in the presence of far-red back-ground light at intensity level 1 (FR 1).

Figure 10. Comparison of ETR(II) light curves measured with dilute Chlorella suspension (300 µg Chl/L) using 440 nm and 625 nm ac-tinic light after transformation of the original PAR scale into a PAR(II) scale (see text). Sigma(II) values of 4.51 and 1.71 nm2 were applied for 440 and 625 nm, respectively. In the calculation of ETR(II)440 and ETR(II)625 Fv/Fm-values of 0.68 and 0.66 were used, respectively. For comparison of the corresponding LC without PAR transforma-tion, see Fig. 11.

Figure 11. Comparison of rel. ETR light curves measured with 440 and 625 nm AL using the original PAR scale. A screenshot of the 440 nm curve has already been shown in Fig. 6.



max)II(Y)II(Y)II(PAR)II(ETR ⋅= (5)

where PAR(II) is the rate of quantum absorption at PS II, Y(II) the effective PS II quantum yield derived from the fluorescence ratio parameter (Fm’-F)/Fm’, Y(II)max the PS II quantum yield in the quasi-dark reference state un-der which Sigma(II)λ was determined and ETR(II) the rate of electron transport expressed in units of elec-trons/(PS II · s).

At very low light intensity, Y(II) approaches Y(II)max, so that Y(II)/Y(II)max = 1 and ETR(II) = PAR(II). This means that there is no loss of PS II efficiency with re-spect to the reference quasi-dark state (all centers open, non-energized, weak far-red background illumination) under which Sigma(II)λ was measured. Y(II)max corre-sponds to the PS II quantum yield of a sample in the same state as given for measurement of k(II) (see e.g. Fig. 5), which equals Fv/Fm. In measurements with algae and cyanobacteria, which may display a relatively high level of PQ-reduction in the dark, it is advisable to meas-ure Fv/Fm in the presence of far-red background light, which oxidizes the PQ-pool and induces the high PS II-efficiency “state I”. Far-red background light is also rou-tinely used for assessment of k(II) and Sigma(II)λ via the O-I1 rise kinetics.

When compared with the common definition of rel. ETR in equation (2), it is apparent that the ETR-factor is con-tained in PAR(II) and that ETR(II) has the dimension of a turnover rate per PS II, whereas rel. ETR commonly has been treated as an electron flux density, i.e. a rate per area, which without information on PS II per area must be considered fictive. In contrast, ETR(II) realistically describes the mean absolute rate of charge separation per PS II in all PS II contained in the 1 mL investigated sam-ple. In practical work with the MULTI-COLOR-PAM, the color and intensity of the applied actinic light as well as the type of investigated samples may be frequently changed. While changes in PAR are accounted for by the PAR-lists, a new Sigma(II)λ determination is required when the type of sample and/or the color are changed.

When the appropriate wavelength- and sample-dependent Sigma(II)λ value is known, the user software supports the transformation of the PAR-lists into corresponding PAR(II)-lists. A practical example of transformation of a PAR-scale into a PAR(II) scale is given in Fig. 10, where the blue curve shows the same ETR Light Curve data as presented in Fig. 5, but presented with a PAR(II) light in-tensity scale. Transformation of PAR-values into PAR(II) values is based on equation 4. The following step-by-step example of calculation applies for PAR = 237 µmol quanta/(m² · s) of 440 nm light using the same Chlorella suspension, with which the data of Figs. 5, 6, 8, 10, and 11 were collected:

s) and II PSper absorbed (quantas644sm

mol10237mol

10022.6m1051.4

smmol10237

mol10022.6nm51.4

PARL)II(Sigma)II(PAR

1

2

623218

2

6232

400440

−

−−

−

=⋅

⋅⋅

⋅⋅⋅=

⋅⋅

⋅⋅

⋅=

⋅⋅=

This calculation is automatically carried out by the PamWin software for all PAR-values at which quantum yields where measured in conjunction with a Light Curve, when the PAR-scale is transformed into a PAR(II) scale.

The corresponding ETR(II)440 value is calculated accord-ing to general equation 5, by entering the values of PAR(II) (with the corresponding unit) and Y(II) = 0.23 for PAR = 237 µmol quanta/(m² · s) as well as Y(II)max = 0.68 for the dark-adapted sample in presence of far-red background light (i.e. condition under which Sigma(II)440 was measured):

s) and II PSper ed transport(electrons s218

68.023.0s644

)II(Y)II(Y)II(PAR)II(ETR

1

1

max440440

−

−

=

⋅=

⋅=

After transformation of PAR into PAR(II), the ETR(II) light curves (LC) measured with different AL colors can be directly compared. The red curve in Fig. 10 shows the corresponding ETR(II)625 LC measured with the same di-lute Chlorella sample, the parameters of which obtained before PAR-transformation were already shown in Figure 7. For comparison, Fig. 11 shows the original LC curves of rel. ETR with 440 and 625 nm AL before transforma-tion of PAR into PAR(II).

Comparison of the data in Figs. 10 and 11 reveals the dis-tinct advantage of transformation of PAR into PAR(II). Whereas before transformation the maximal rel. values differed by a factor of 2.58, after transformation the ETR(II)440 and ETR(II)625 are practically equal. This may be considered strong support for the validity of Sigma(II)λ determination via O-I1 measurements with the MULTI-COLOR-PAM and its analysis by the O-I1 Fit approach.

When information on PS II concentration is available, this may serve to derive an estimate of absolute O2 evo-lution rate with the common unit of µmol O2/(mg Chl · h) from ETR(II). As will be shown below, a very simple general equation can be applied for this calculation. For understanding the background, however, reading the fol-lowing points may be useful, with step-by-step calcula-tions specifically for the experiment of Fig. 10 with PAR = 237 μmol quanta/(m² · s) of 440 nm light, for which calculation of PAR(II) and ETR(II) already was outlined above:



1) In the MULTI-COLOR-PAM cuvette an area of 100 mm2 is illuminated. As measurements are carried out with highly dilute samples, it may be assumed that the incident PAR (measured either with the planar sensor MQS-B at the exit of the 10 x 10 mm Perspex light guide rod or with the spherical sensor US-SQS/B within the 10 x 10 mm cuvette) is effective throughout the whole sample. Hence, the number of PS II units within 1 mL of the investigated suspen-sion is decisive for the absolute rate.

2) The overall Chl content of the Chlorella suspension equals 300 µg Chl/L, which corresponds to 3 · 10-7 g Chl/mL.

3) This amounts to 3.333 · 10-10 mol Chl/mL, assuming a molecular weight of 900 g/mol for Chl.

4) As one mol contains 6.022 · 1023 molecules, this cor-responds to 2.01 · 1014 molecules of Chl/mL.

5) Assuming a photosynthetic unit (PSU) size of 1000 Chl molecules per electron transport chain (including one PS II each), it follows that about 2.01 · 1011 PS II are contained in the illuminated 1 mL suspension. Based on this information, a volume related electron transport rate can be calculated from

ETR(II)440=218 electrons/(PS II · s):

6) Electrons transported by all PS II contained in 1 mL suspension:

smLelectrons104381001.2218re 1111

⋅⋅=⋅⋅=−

and, as 4 electrons are transported by PS II for evolu-tion of one molecule O2 and 1 h = 3600 s, the corre-sponding volume related O2 evolution rate amounts to

hmLOmol10654.0

10022.61094.3

hmLOmolecules1094.3

4360010438rO

2723

16

216112

⋅⋅=

⋅⋅

=

⋅⋅⋅=⋅⋅=

−

which is the absolute rate at which the O2 concentra-tion in the sample increases with time, as it can be also directly measured with an O2-electrode.

7) In physiological work, photosynthetic electron trans-port rate often is expressed in µmol O2/(mg Chl · h), i.e. Chl mass related rO2. For transformation into these units it has to be considered that 1 mL of 300 µg Chl/L suspension contains 3 · 10-4 mg Chl

hChlmgOµmol218

hmLOmol

10310654.0rO 22

4

7

2 ⋅=

⋅⋅⋅

= −

−

It may be noted that the numerical value of the rate ex-pressed in µmol O2/(mg Chl · h) is identical to that ex-pressed in electrons/(PS II · s). This simple relationship, however, is valid only with the specific assumptions made above, i.e. a functional photosynthetic unit (PSU)

size of 1000 Chl/PSU and a molecular weight of Chl of 900 g/mol.

The absolute rate of O2-evolution in units of mmol O2/(mg Chl · s) can be derived from ETR(II) by the following general equation:

)Chl(M)O(nePSU)II(ETRrO

22 ⋅⋅=

(6)

where M(Chl) is the molecular weight of Chl and ne(O2) the number of electrons required for evolution of 1 mole-cule of O2 (normally assumed to be 4). The absolute rate in the common units of µmol O2/(mg Chl · h) is obtained by multiplication with 3600 · 1000. As one can see, the identical numerical values for ETR(II) in electrons/(PS II · s) and rO2 in µmol O2/(mg Chl · h) is owed to the co-incidence that the molecular weight of Chl times ne(O2)=4 equals the number of seconds in one hour.

3.4 Leaf measurements For measurements with leaves a special leaf holder is available, measuring fluorescence from the leaf surface. As high chlorophyll content is unavoidable in the case of leaves, pronounced light intensity gradients are the rule, even with green light, resulting in heterogeneous photo-synthetic responses in different cell layers at varying depths from the illuminated surface (see e.g. Vogelmann 1993; Schreiber et al. 1996). While it is generally impos-sible to measure the overall photosynthetic response of a leaf via chlorophyll fluorescence, the MULTI-COLOR-PAM allows for relatively homogenous responses from the top cell layers at upper and lower leaf surfaces. For this purpose, two aspects are essential:

a) Use of 440 nm pulse-modulated measuring light (ML), which is almost completely absorbed within the top cell layer.

b) Use of a short-pass filter (< 710 nm) in front of the de-tector, so that selectively short-wavelength fluorescence is measured, which almost completely originates from the top cell layer, as most of the short-wavelength fluores-cence that is excited within deeper layers (despite of the use of 440 nm ML) will be reabsorbed by the chloro-plasts in the upper cell layers.

Figs. 12 and 13 show fluorescence responses (F<710 nm) measured from the upper (adaxial) and lower (abaxial) surfaces of the same ivy leaf using 440 nm ML. In the experiment of Fig. 12 the dark-adapted leaf was illumi-nated with continuous 440 nm AL starting at time 0, re-sulting in typical dark-light induction curves (Kautsky ef-fect).

The dark-light induction kinetics of upper and lower leaf sides differ considerably, although the same intensity of AL is applied (see also Schreiber et al. 1977). While a de-tailed discussion of the differences would be out of the scope of the present communication, it may be noted that the response of the lower leaf side is more pronounced,



just as if the applied PAR were higher. In principle, this could be either due to a larger functional cross-section of PS II, Sigma(II)440, or to a smaller acceptor pool size in the shade-adapted lower leaf side. Fig. 13 shows O-I1 rise curves measured from upper (left) and lower (right)

sides of the same ivy leaf. As already is obvious by visual comparison, the initial slope is significantly higher at the lower than at the upper leaf side. Upon fitting of the two curves 32% larger values of k(II) and Sigma(II)440 are suggested for the lower vs. the upper leaf surface.

Figure 12. Dark-light induction curves (Kautsky effect) of ivy leaf measured from upper (left curve) and lower (right curve) sides of the same leaf. 440 nm ML switched on 100 ms before onset of actinic illumination (118 µmol quanta m-2 s-1 440 nm) at time 0. Fluorescence detection at wavelengths < 710nm.

Figure 13. O-I1 rise curves induced by strong actinic light (440 nm multiple turnover pulse, MT) at upper (left curve) and lower (right curve) sides of the same ivy leaf. The two curves are normalized at the I1-level. Dashed yellow lines indicate the Fo-level (O) assessed during a 50 µs period preceding onset of at time zero and the I1-level that is determined with the help of a saturating multi-color single-turnover pulse (ST) triggered 1ms after onset of MT. The slope of the relaxation kinetics is extrapolated to the end of the 50 µs ST. The black line represents the O-I1 fit curve based on a PS II model which incorporates energy transfer between PS II units and reoxidation of the primary PS II acceptor QA (see text to Fig. 8).

Lower leaf side

Lower leaf side

Upper leaf side

Upper leaf side



4 Single turnover pulses, ST

4.1 General features

The same multi-color LED array that generates AL, is also employed for generating single turnover light pulses, ST. The user may choose between ST with one of 6 dif-ferent colors (440, 480, 540, 590, 625 nm and white) and “multi-color ST”, with all of these 6 colors being applied simultaneously. A multi-color ST at 50 µs width nor-mally is saturating, which is not necessarily true for sin-gle color ST. For Fast Kinetics recordings, ST can be programmed with 2.5 to 50 µs width.

The duration of single turnover pulses is so short that not more than one charge separation per PS II reaction center is possible, as the reoxidation of the primary PS II accep-tor QA by the secondary acceptor QB is characterized by time constants in the order of 200-600 µs. ST-induced QA reduction and the ensuing reoxidation kinetics can be readily measured in the fast kinetics mode of the MULTI-COLOR-PAM (Fig. 14). In many applications it is essen-tial that the applied ST pulses are saturating, which means that all PS II reaction centers turn over once and, hence, a maximal increase of fluorescence yield is ob-tained.

4.2 ST-efficiency and exponential saturation characteristics

The ST-efficiency, i.e. the extent of ST-induced fluores-cence increase depends on a number of parameters, namely:

• Choice of ST color or Multi-Color

• Selected ST width

• Chlorophyll content of the sample

• PS II antenna composition of the sample

• Filter in front of the photodetector (particularly with dense samples, like leaves)

• Choice of ML color (particularly with dense samples, like leaves).

The MULTI-COLOR-PAM displays exceptionally high sensitivity, so that rather low chlorophyll content is feasi-ble, which favors high ST-efficiency (practically no shad-ing). Measurements at low Chl content are also advanta-geous, because light-intensity gradients are avoided. This is particularly important, for evaluation of the light-saturation characteristics and assessment of potential het-erogeneities in PS II antenna size (Melis 1991, Lavergne and Briantais 1996).

The data presented in Figs.14 and 15 were obtained with a dilute suspension of Chlorella vulgaris at a Chl a con-tent of 400 µg/L (green color just visible). Fast relaxation kinetics in response to multi-color ST pulses of various widths (5, 10, 20 and 50 µs) are shown in Fig. 14. The

sample was continuously illuminated with far-red back-ground light in order to establish defined conditions in terms of PS II acceptor side oxidation and pigment state 1.

Analogous data were collected for additional ST-widths (i.e. 2.5, 7.5, 12.5, 15, 30 and 40 µs, kinetics not shown) and a corresponding set of data was collected using 440 nm ST pulses (kinetics not shown). These measure-ments were carried out with the help of a preprogrammed script-file (STwidth_relax.prg), which automatically opens the corresponding Fast Trigger files for measuring the ST-induced fluorescence increase followed by the re-laxation kinetics. Under the given conditions with multi-color ST pulses close to saturation is already reached with 20 µs width, whereas 50 µs width is required in the case of 440 nm ST pulses.

The saturation curves of the ST-induced fluorescence in-crease (ΔF) as a function of ST-width are presented in Fig. 15 for both multi-color and 440 nm ST pulses. As the pulses display steep on/off slopes, ST width is pro-portional to ST pulse energy and, hence, the plot of the ST-induced ΔF vs. ST width corresponds to classical flash energy saturation curves (Falkowski et al. 1986, Mauzerall and Greenbaum 1989). From the data in Fig. 15 it can be estimated that with a dilute Chlorella suspension the multi-color ST are about 2.3 times more effective in terms of PS II turnover than the 440 nm ST. In the given example, even a single-color (440 nm) 50 µs ST is saturating. While the 440 nm LED chips amount to only about 14% of the overall number of actinic chips, the 440 nm light is particularly well absorbed in Chlor-ella.

4.3 ST-pulse intensity and functional PS II cross-section Sigma(II)λ

The light intensity corresponding to a 440 nm ST pulse can be deduced by comparison with the response of the same sample to a 440 nm AL or MT pulse (see section below), the intensity of which can be directly measured. The ΔF induced by a 50µs 440 nm AL pulse of 3613 µmol quanta/(m² · s) matches the ΔF induced by a 3.6 µs 440 nm ST. Ignoring QA

- reoxidation and assuming that the initial rise of ΔF vs. ST-width is linear, in first ap-proximation it may be assumed that the quantum flux density in the 440 nm ST pulse is 50/3.6=13.88 times higher than in the 440 nm AL pulse. Then 440 nm ST-pulse intensity amounts to 13.88 · 3613 = 50180 µmol quanta/(m² · s) and a 50µs ST pulse contains 1.51 · 1018 quanta/m² (Avogadro’s constant L = 6.022 · 1023 mol-1). Based on this information and on the 440 nm ST satura-tion curve in Fig. 15, the MULTI-COLOR-PAM provides an alternative method for estimation of the wavelength-dependent functional PS II absorption cross-section, Sigma(II)λ (for determination by analysis of O-I1 rise ki-



netics, see section 3.2). An example of application of this method is described here in some detail, in order to show the basic equivalence between the classical “single-turnover flash saturation” and the “O-I1 fit” approaches.

The light-induced closure of PS II reaction centers by ST-pulses with increasing pulse widths in first approximation can be described assuming simple exponential saturation characteristics according to:

tPARL)II(Sigma

sat

440e1FΔFΔ ⋅⋅⋅−−=

(7)

where ΔF is the increase of fluorescence yield with the ST-width corresponding to time t (s), ΔFsat the maximal increase of fluorescence yield with a saturating ST, Sigma(II)440 the PS II absorption cross-section for 440 nm quanta (m²), L the Avogadro constant (mol-1) and PAR the quantum flux density in mol/(m² · s) during the ST.

An average of 63% of PS II centers are closed when Sigma(II)440 · PAR · L · t = 1 and, hence, ΔF = ΔFsat (1-1/e), i.e. when ΔF is 63% of the saturated ΔF. From the 440 nm ST saturation curve in Fig. 15 it can be estimated that this response is obtained with a 15.17 µs ST, which corresponds to 0.474 · 1018 quanta/m². Hence, for one 440 nm quantum the effective cross-section area is 2.18 nm2, which is Sigma(II)440. This value of Sigma(II)440 is significantly lower than the 4.51 nm2 de-termined for Chlorella via analysis of the O-I1 rise (see section 3.2). It should be noted that in the ST-based ap-proximation of Sigma(II)λ, linearity between ΔF and QA

- is assumed, whereas in reality the relationship is non-

linear due to energy transfer between PS II units (Joliot and Joliot 1967). Furthermore, reoxidation of QA

- during the ST and the ensuing time period before measurement of fluorescence yield is not taken into account, which re-sults in underestimation of Sigma(II). Last but not least also an intrinsic inefficiency of charge separation at very high light intensities (Rappaport et al. 2007) could be re-sponsible for a lower value of Sigma(II) when measured by the ST-saturation approach as compared to the O-I1 fit approach.

Figure 14. Multi-color ST-induced fluorescence increase in dilute Chlorella-suspension (400 µg Chl/L) at ST-widths of 5, 10, 20 and 50 µs. Fluorescence excitation with #3ML (540 nm). Continuous far-red background illumination (FR 9). Relaxation curves displayed with Log time scale.

Figure 15. ST-induced fluorescence increase in dilute suspension of Chlorella vulgaris as function of the width of ST pulses applied either with the single color #1 (440 nm) or with the combination of all colors (Multi-Color). Examples of Multi-Color ST-induced relaxation kinetics are shown in Fig.14.

Figure 16. Fluorescence increases induced by 50 µs ST pulses of various colors in dilute suspensions of Chlorella and Synechocystis in relation to the saturating effect of a 50 µs multi-color ST (100%). Fluorescence excitation with 540nm. Continuous far-red background illumination (FR 9).



Hence, although the MULTI-COLOR-PAM provides the means to estimate Sigma(II) via classical flash saturation curves, measurements of the O-I1 rise kinetics and deter-mination of Sigma(II) with the help of the O-I1 fitting routine provided by the PamWin software are not only more rapid and convenient, but also more reliable for 3 major reasons:

• relatively moderate actinic intensity is used, thus minimizing inefficiency caused by extremely high quantum flux densities;

• the connectivity/sigmoidicity parameter J is fitted, so that non-linearity between ΔF and QA

- can be ac-counted for;

• the applied PS II model takes account of QA- oxida-

tion by QB.

4.4 Relative ST pulse effects of the various colors in Chlorella and Synechocystis

The fluorescence increases (ΔF) induced by variously colored ST pulses in differently pigmented organisms like Chlorella and Synechocystis depend on the ST pulse

energy (quanta·m²) and the wavelength-dependent func-tional cross-section of PS II, Sigma(II)λ, of the investi-gated sample. While for technical reasons saturation can-not be reached with all single colors, this is always possi-ble using a Multi-Color ST. Hence, the effect of variously colored ST pulses can be quantified relative to the satu-rated effect induced by a Multi-Color ST (see Fig. 16).

It may be noted that there is some similarity between the spectra of ST-induced ΔF in Fig. 16, of Fo/PAR in Fig. 2 and of Sigma(II) in Fig. 9. This is true, although the ST-intensities of the various colors differ from each other. In first approximation, the relative differences may be esti-mated from the PAR-values at maximal AL-intensity set-tings presented in Table 3. In this way, spectra of ΔF(ST)/PAR can be derived (not shown) that provide similar information as Sigma(II) spectra. The practical value of such spectra decisively depends on the accuracy, with which the ST intensities of the various colors can be measured.

5 Multiple turnover pulses, MT and saturation pulses, SP

5.1 General features The same multi-color LED array that generates AL and ST, is also employed for generating “Multiple Turnover” light pulses, MT and Saturation Pulses, SP. There is no principal difference between AL and MT/SP, except for three features:

a) Maximal MT/SP intensities can be more than twice higher than the corresponding maximal AL intensities.

b) Maximal MT/SP width is limited to 800 ms, whereas AL can be applied for an unlimited time.

c) The increase of MT/SP intensity with increasing set-tings is close to linear, whereas it is exponential in the case of AL.

The user may choose between one of 6 different colors (440, 480, 540, 590, 625 nm and white), which at the same time apply for AL, MT/SP and ST.

While MT pulses and SP do not differ physically, they serve different functions in PAM fluorimetry. MT pulses are mostly used with Fast Kinetics recordings. They can be programmed in Fast Trigger files. For maximal kinetic resolution normally switching to MF-max (100 or 200 kHz) is triggered. On the other hand, SP are used for Saturation Pulse quenching analysis, e.g. in conjunction with Slow Kinetics and Light Curve recordings. With every SP automatically Y(II) and other fluorescence based parameters are calculated, which is not the case with MT. During an SP the frequency of pulse-modulated ML is automatically switched to 10 kHz.

Major differences between MT and ST are:

a) MT can be applied at largely different intensities, in analogy to AL. In contrast, ST are always applied at maximal intensity, with their effect being controlled by the ST-width.

b) Maximal MT-width is 800 ms, whereas ST-width is limited to a maximal value of 50µs. The minimal value is 2.5 µs.

c) With ST in contrast to MT all colors can be applied simultaneously.

For each color the user may choose between 20 MT in-tensity settings, corresponding to defined LED currents and PAR values at the exit of the Perspex light guide of the emitter unit. For standard applications default cur-rent/PAR lists (default_MC.par and default_leaf.par) with close to linearly increasing intensity steps are provided.

The default lists can be readily modified by the user and saved in new par-files. LED current values can be pro-grammed in 255 steps.

The PAR values defined in the default lists were meas-ured with the help of the automated <Measure PAR Lists> routine using the US-SQS/B and MQS/B sensors (Walz) for suspension cuvette and leaf holder, respec-tively. These sensors can resolve the kinetics of short light pulses and determine the PAR during the first milli-seconds of the pulse. This is important, as the LED-chips unavoidably heat up with high current pulses, which leads to significant drops of intensity within the 10-100 ms time range (particularly in the case of the 590 and 625 nm LEDs, which display a relatively high tem-perature coefficient). Accurate PAR-measurements are essential in conjunction with determination of the wave-length dependent functional cross-section of PS II,



Sigma(II)λ (see section 3.3). As the O-I1 measurement is terminated after 1-2 ms, it is not disturbed by the heating of the LED-chips during an MT pulse.

Depending on the selected width, MT/SP in contrast to ST pulses can induce multiple turnovers at PS II reaction centers and thus cause full reduction not only of the pri-mary acceptor QA, but also of QB and the plastoquinone acceptor pool, PQ, which is required for assessment of maximal fluorescence yield Fm or Fm’(see Schreiber 2004). An SP serves the function of inducing full reduc-tion of the acceptor side, for which purpose the color, in-tensity and width have to be appropriately selected. The SP kinetics, which are automatically saved under Fast Kinetics, indicate whether the selected parameters are appropriate. Essential criteria are:

• A plateau level is reached.

• The intensity is not much higher than required to reach maximal amplitude. In particular, the intensity should be decreased, if the signal transiently jumps to a somewhat higher value when the SP/MT is over. This phenomenon reflects donor-side dependent quenching.

• The width is not much longer than required to reach a plateau, in order to minimize the dose of potentially harmful strong light.

The time resolution of the SP kinetics is not high enough for kinetic analysis of the polyphasic fluorescence rise upon onset of saturating light (O-I1-I2-P curve) which,

however, can be routinely measured with the help of MT pulses and preprogrammed Fast Trigger files.

5.2 Polyphasic fluorescence rise, O-I1-I2-P Fig. 17 shows a typical example of a polyphasic fluores-cence rise measured with a dark-adapted ivy leaf using 440 nm ML and a 440 nm MT pulse of maximal intensity (MT20). Using a logarithmic time scale the three con-secutive rise phases in the ranges of 100µs (O-I1), 10ms (I1-I2) and 100 ms (I2-P) can be displayed with distinct plateau regions in between, corresponding to the inter-mediate levels I1 and I2. The clear-cut separation of the various phases is due to the following features provided by the MULTI-COLOR-PAM:

• Use of a very high light intensity, which assures full reduction of the primary acceptor QA during the pho-tochemical O-I1 phase before onset of the first ther-mal phase (I1-I2).

• Measuring fluorescence from the surface in the case of leaves or with dilute suspensions in the case of unicellular algae or cyanobacteria.

• In the case of leaves, use of 440 nm ML for almost selective excitation of the top cell layer, where irradi-ance is close to homogenous;

• In the case of leaves, detection of short-wavelength fluorescence (<710 nm), so that selective assessment of the top cell layer is further enhanced.

Figure 17. Polyphasic rise of fluorescence yield (F<710nm) upon onset of saturating light in dark-adapted ivy leaf (upper surface). Characteris-tic fluorescence levels O-I1-I2-P are indicated. Use of 440 nm ML and 440 nm multiple turnover pulse (MT 20). Display with logarithmic time scale.



Elimination of F>710 nm also enhances the ratio of PS II/PS I fluorescence in the measurement of PS II quantum yield, Fv/Fm and Y(II), as well as of the derived electron transport rate, ETR(II), improving their accuracy

Measurements of the O-I1-I2-P rise kinetics in suspen-sions of unicellular algae and cyanobacteria are compli-cated by the fact that in these organisms during dark-adaptation more or less strong electron flow from re-duced stroma components to plastoquinone (PQ) takes place, so that the redox state of the PQ-pool is undefined. The latter is problematic for three reasons:

a) Reduced PQ is in equilibrium with reduced QB (QB-

and QB2-) and QA

-, which may lead to overestimation of Fo and acceleration of the O-I1 rise.

b) PQ reduction affects the quenching of fluorescence yield at the I1-level (apparent PQ-quenching, see e.g. Schreiber 2004), thus causing uncontrolled variability of I1.

c) PQ reduction triggers a state transition from state 1 to state 2, which may affect the functional cross-section of PS II.

In applications, where a defined state of the PS II accep-tor side is essential (e.g. for determination of wavelength dependent functional cross-section of PS II, see sections 3.2 and 3.3), these problems can be avoided by far-red (FR) background light, which is provided by the MULTI-COLOR-PAM.

The multi-color Chip-on-board LED-array features a sin-gle 1x1mm far-red power-chip with peak emission at 725 nm and 30 nm half-band width. Wavelengths below 700 nm are suppressed by a long-pass filter. The user may choose between 20 FR intensity settings which are defined in the corresponding current/PAR list. In the de-

fault current/PAR list (default_MC.par) the same current values are used for FR as for 625 nm AL (see Fig. 3), i.e. currents and intensities increase exponentially with in-creasing settings. The default list can be readily modified by the user and the new list saved in a new par-file. LED current values can be programmed in 255 steps.

FR plays an important role in photosynthesis research and in PAM work in particular, as it is much better ab-sorbed by PS I than by PS II. FR illumination results in oxidation of P700 and consequently of the intersystem electron transport chain, including the PQ-pool. At a given FR intensity, the relative extent of FR-induced oxi-dation depends on the rate at which PQ is reduced either by PS II activity, cyclic PS I activity or electron donation by stroma components in the dark.

The latter mechanism is particularly pronounced in algae and cyanobacteria, where dark reduction of the PQ-pool leads to transition from state 1 to state 2, with the latter being characterized by decreased PS II excitation. The MULTI-COLOR-PAM is ideally suited for investigations of this important regulatory mechanism. A separate PAN article on measurements of reversible state 1-state 2 tran-sitions and assessment of the wavelength-dependent functional cross-section of PS II is in preparation.

While FR is much more effective in excitation of PS I relative to PS II, which indirectly affects fluorescence yield via the redox state of the intersystem electron trans-port chain, its direct effect on PS II can also be signifi-cant, particularly with respect to so-called “inactive PS II” (Lavergne and Leci 1993). The MULTI-COLOR-PAM has opened the way for a profound analysis of this type of PS II heterogeneity, which will be outlined in a separate PAN article.

6 References

Björkman O and Demmig B (1984) Photon yield of O2-evolution and chloroplast fluorescence characteristics at 77K among vascular plants of diverse origins. Planta 170: 489-504

Chylla RA, Garab G and Whitmarsh J (1987) Evidence for slow turnover in a fraction of Photosystem II com-plexes in thylakoid membranes. Biochim Biophys Acta 894: 562-571

Eilers PHC and Peeters JCH (1988) A model for the rela-tionship between light intensity and the rate of photosyn-thesis in phytoplankton. Ecological Modelling 42: 199-215

Falkowski PG, Wyman K, Ley AC and Mauzerall DC (1986) Relationship of steady-state photosynthesis to fluorescence in eucaryotic algae. Biochim Biophy Acta 849: 183-192

Gilbert M, Domin A, Becker A and Wilhelm C (2000a) Estimation of primary productivity by chlorophyll a in vivo fluorescence in freshwater phytoplankton. Photosyn-thetica 38(1): 111-126

Gilbert M, Wilhelm C and Richter M (2000b) Bio-optical modelling of oxygen evolution using in vivo fluores-cence: Comparison of measured and calculated photosyn-thesis/irradiance (P-I) curves in four representative phytoplankton species. J Plant Physiol 157: 307-314

Koblizek M, Kaftan D and Nedbal L (2001) On the rela-tionship between the non-photochemical quenching of the chlorophyll fluorescence and the Photosystem II light harvesting efficiency. A repetitive flash fluorescence in-duction study. Photosynth Res 68: 141-152



Kolber ZS, Prasil O and Falkowski PG (1998) Measure-ment of variable chlorophyll fluorescence using fast repe-tition rate techniques: defining methodology and experi-mental protocols. Biochim Biophys Acta 1367: 88-106

Lavergne J and Briantais J-M (1996) Photosystem II het-erogeneity. In: Ort DR and Yocum CF (ed): Oxygenic Photosynthesis: The Light Reactions. pp 265-287. Klu-wer Academic Publishers, Dordrecht-Boston-London

Lavergne J and Leci E (1993) Properties of inactive PS II centers. Photosynth Res 38: 323-343

Lavergne J and Trissl H-W (1995) Theory of fluores-cence induction in Photosystem II: Derivation of analyti-cal expressions in a model including exciton-radical-pair equilibrium and restricted energy transfer between photo-synthetic units. Biophys J 68: 2474-2492

Mauzerall D and Greenbaum NL (1989) The absolute size of a photosynthetic unit. Biochim Biophys Acta 974: 119-140

Melis A (1991) Dynamics of photosynthetic membrane composition and function. Biochim Biophys Acta 1058: 87-106

Nedbal L, Trtílek M and Kaftan D (1999) Flash fluores-cence induction: a novel method to study regulation of Photosystem II. J Photochem Photobiol B: Biol 48: 154-157

Platt T, Gallegos CL and Harrison WG (1980) Photoin-hibition of photosynthesis in natural assemblages of ma-rine phytoplankton. J Marine Res 38: 687-701

Rappaport F, Béal D, Joliot A and Joliot P (2007) On the advantage of using green light to study fluorescence yield changes in leaves. Biochim Biophys Acta 1767: 56-67

Sakshaug E, Bricaud A, Dandonneau Y, Falkowski PG, Kiefer DA, Legendre L, Morel A, Parslow J and Takaha-shi M (1997) Parameters of photosynthesis: definitions, theory and interpretation of results. J Plankton Res 19 (11): 1637-1670

Samson G, Prasil O and Yaakoubd B (1999) Photo-chemical and thermal phases of chlorophyll a fluores-cence. Photosynthetica 37(2): 163-182

Schreiber U (2004) Pulse-Amplitude (PAM) fluorometry and saturation pulse method. In: Papageorgiou G and Govindjee (eds) Chlorophyll fluorescence: A signature of Photosynthesis, pp 279-319. Kluwer Academic Publish-ers, Dordrecht, The Netherlands

Schreiber U, Bilger W and Neubauer C (1994) Chloro-phyll fluorescence as a non-intrusive indicator for rapid assessment of in vivo photosynthesis. Ecological Studies 100: 49-70

Schreiber U, Fink R and Vidaver W (1977) Fluorescence induction in whole leaves: Differentiation between the two leaf sides and adaptation to different light regimes. Planta 133: 121-129

Schreiber U, Kühl M, Klimant I and Reising H (1996) Measurement of chlorophyll fluorescence within leaves using a modified PAM fluorometer with a fiber-optic mi-croprobe. Photosynth Res 47: 103-109

Stirbet A, Govindjee (2011) On the relation between the Kautsky effect (chlorophyll a fluorescence induction) and Photosystem II: Basics and applications of the OJIP fluo-rescence transient. J Photochem Photobiol B: Biol 104: 236-257

Strasser, RJ, Tsimilli-Michael M and Srivastava A (2004) Analysis of the chlorophyll a fluorescence transient. In: Papageorgiou G and Govindjee (eds) Chlorophyll fluo-rescence: A signature of Photosynthesis, pp 321-362. Kluwer Academic Publishers, Dordrecht, The Nether-lands

Vogelmann TC (1993) Plant tissue optics. Ann Rev Plant Physiol Plant Mol Biol 44: 231-251

high-end chlorophyll fluorescence analysis with the multi

Documents